Abstract
TGF-β1 is a disulfide-bonded homodimeric protein produced by platelets and other cells that plays a role in many physiologic and pathologic processes. TGF-β1 is secreted as an inactive large latent complex (LLC) comprised of TGF-β1, latency-associated peptide, and latent TGF-β binding protein 1. We previously demonstrated that shear force can activate LLC and that thiol-disulfide exchange contributes to the process. We have now investigated the role of thiol isomerases in the activation of LLC in platelet releasates (PR) and recombinant LLC. The wasp venom peptide mastoparan, which inhibits the chaperone activity of PDI, inhibited stirring- and shear-induced activation of latent TGF-β1 by 90 and 75% respectively. To identify the proteins that bind to mastoparan either directly or indirectly, PR were chromatographed on a mastoparan affinity column. Latent TGF-β binding protein 1, latency-associated peptide, TGF-β1, clusterin, von Willebrand factor, multimerin-1, protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72 eluted specifically from the column. Anti-PDI RL90 attenuated the inhibitory effect of mastoparan on LLC activation. Furthermore, reduced PDI inhibited activation of PR LLC, whereas oxidized PDI had no effect. We conclude that thiol isomerases and thiol-disulfide exchange contribute to TGF-β1 activation and identify a number of molecules that may participate in the process.
Highlights
We previously demonstrated that shear-induced TGF-1 activation involves thiol-disulfide exchange
Mastoparan Does Not Disrupt the Covalent Association of latent TGF-1 binding protein 1 (LTBP-1) and latency-associated peptide (LAP) That Is Necessary for Shear-induced Activation of TGF-1—Because we previously found that LTBP-1 must be covalently bound to LAP for TGF-1 to be activatable by shear [12], we assessed whether mastoparan affected the covalent association of LTBP-1 with LAP
We extended our previous findings by showing that stirring could activate recombinant large latent complex (LLC)
Summary
We previously demonstrated that shear-induced TGF-1 activation involves thiol-disulfide exchange. Results: The thiol isomerase binding peptide mastoparan and protein disulfide isomerase inhibit shear-induced TGF-1 activation. We previously demonstrated that shear force can activate TGF-1 released from platelets in vitro and that TSP-1 partially contributes to the activation process [12, 13]. We found that stirring or shear decreased the ability of thiol-reactive agents to label proteins in platelet releasates, including TGF-1 and TSP-1, suggesting an association between thiol oxidation and TGF-1 activation. A role for thiol-disulfide exchange in TGF-1 activation was further supported by our finding that multiple thiol-reactive agents inhibited stirring-induced activation. Because thiol isomerases play an important role in catalyzing thiol-disulfide exchange, in this study, we assessed the effects of mastoparan, a peptide that inhibits thiol isomerase chaperone activity, and both oxidized and reduced PDI on shear-dependent TGF-1 activation
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